Simultaneous self-renewing and asymmetric cell divisions are a hallmark of stem cells. We can now provide a mechanism for how the germline stem cells of Drosophila accomplish this feat. Dpp signaling in the stem cell niche blocks transcription of the key cystoblast differentiation factor bam in the stem cell. It is not clear, however, why the cystoblast does not also respond to Dpp signaling. The goal of the work proposed here seeks a deeper and mechanistic understanding of additional niche signaling that might explain the differential responses of stem cell and cystoblast. Specific Aim 1. MECHANISMS MAINTAINING GERMLINE STEM CELLS (GSCs). Determine roles of multiple GSC maintenance factors using tools developed to assay bam transcriptional activation and to manipulate dpp-signaling levels. Determine if other niche signaling pathways act through dpp signaling or independent of dpp signaling. Specific Aim 2. MECHANISMS THAT DRIVE CYSTOBLAST (CB) DIFFERENTIATION. Test hypothesis that arises from preliminary results - pre-CBs express dpp signaling antagonist. Determine the identity, time of action and regulation of antagonist. Specific Aim 3. MOLECULAR BIOLOGY OF THE BAM GENE SILENCING MECHANISM. Fully characterize the cis-active elements and trans-acting factors involved in the silencing mechanism. Test if Schnurri works with Mad to quench bam transcription. The experimental approaches employ mitotic clone tests of the relevance of shn for bam expression patterns and transgenic constructs to test predictions of the mechanisms regulating silencer activity. Specific Aim 4. GENETIC SCREENS TO IDENTIFY GSC AND CB DIFFERENTIATION FACTORS. Preliminary results support two genetic approaches to identify new GSC and CB differentiation factors. Gene misexpression from P-element insertions can identify genes that disrupt GSC or CB fates when expressed from a strong germ cell promoter. A "sensitized" bam genotype can identify genes that can reduce (E(bam) or increase (Su(bam) bam activity when made heterozygous.